Antibodies have received attention as drugs because of having high stability in plasma and producing few adverse reactions. Among others, many IgG-type antibody drugs have been launched, and a large number of antibody drugs are also currently under development (Non Patent Literatures 1 and 2). Meanwhile, various techniques have been developed as techniques applicable to second generation antibody drugs. For example, techniques of improving effector functions, the ability to bind to antigens, pharmacokinetics, or stability or reducing immunogenic risks have been reported (Non Patent Literature 3).
In recent years, multispecific antibodies such as bispecific antibodies (BsAbs) have received attention as one of methods for highly functionalizing antibodies. BsAb is one kind of multivalent antibody capable of binding to two types of antigens by possessing, in one molecule, sites capable of binding to two different antigenic determinants (epitopes).
BsAb typically comprises two types of H chains and two types of L chains. A problem associated with the production of BsAb is that when these H chains and L chains are transferred to one cell and expressed therein, immunoglobulin H chains are combined with immunoglobulin L chains at random, possibly producing 10 different types of antibody molecules (Non Patent Literature 4 and Patent Literature 1). Of these 10 types of antibodies produced, an antibody having desired bispecificity is only one type of antibody constituted by a combination of two H chain-L chain pairs differing in binding specificity in which each H chain is correctly combined with each L chain.
Methods for efficiently heterodimerizing produced H chains are known as methods to solve such a problem. Examples of such known methods include: a method which involves introducing structures sterically complementary to each other to two CH3 domains (Non Patent Literature 5 and Patent Literature 2); a method which exploits the properties of IgG and IgA CH3 domains of not binding to each other and involves converting two CH3 domains only to a desired heterodimer by interdigitating an IgG-derived sequence and an IgA-derived sequence (SEEDbodies: Non Patent Literature 6); and a method which involves promoting heterodimerization through the use of the charge interaction between two H chains by introducing a mutation to their CH3 domains (Patent Literature 3).
Unfortunately, the H chains produced by these methods still may pair with wrong L chains. Accordingly, methods for producing a multispecific antibody having common L chains while promoting the heterodimerization of H chains have been reported. Examples of known methods for obtaining common L chains include: a method for obtaining common L chains by preparing a library of L chains, sequentially combining each L chain of the library with H chains of two antibodies, and screening for an antibody capable of binding to their respective antigens (Patent Literature 4); a method which involves obtaining antibodies binding to different antigens from an antibody library having a limited repertoire of L chains, and selecting antibodies having identical L chains from among the obtained antibodies (Non Patent Literature 7 and Patent Literature 1); a method which involves preparing chimeric L chains by the shuffling of CDRs of two types of antibody L chains, and screening for common L chains capable of binding to both antigens (Non Patent Literature 8); a method for obtaining an antibody having common L chains by immunizing a transgenic mouse harboring a particular L chain gene (Patent Literatures 5 and 6); and a method for obtaining an antibody having common L chains by obtaining antibodies binding to different antigens from an antibody library containing a particular L chain gene and having diverse H chains (Non Patent Literature 14).
Alternative examples of such known methods include: a method for promoting selective heterodimerization by altering H chain and L chain constant regions (Patent Literature 3); a method for preparing only a desired heterodimer by H chain variable region/L chain variable region (VH/VL) or H chain constant region CH1/L chain constant region (CH1/CL) crossover (Crossmab: Patent Literature 7); and a method for preparing a bispecific antibody by preparing two types of antibodies, followed by in vitro disulfide bond isomerization (DuoBody: Patent Literature 8).
Furthermore, a method which involves obtaining antibodies against various antigens using a common H chain library and an L chain library, and then preparing a bispecific antibody from common H chains and two types of L chains (κ chain and λ chain) is known (Kappa-Lambda Body: Patent Literature 11) in relation to a method for obtaining common H chains.
Alternatively, antibodies that recognize different epitopes on the same antigen are obtained and may be used in a bispecific antibody (particularly, biparatopic antibody). Upon antigen binding of the biparatopic antibody, even single antigens can be cross-linked by the biparatopic antibody to form an immune complex (IC). The in vivo formation of this immune complex is expected to offer the rapid clearance of the immune complex from blood (Patent Literature 9).
Meanwhile, phage display technology is increasingly adopted widely as one of methods for obtaining antigen-binding molecules. The phage display technology is a technique of displaying, for example, H chain variable regions and L chain variable regions of antibodies on the particles of bacteriophages. A population of many bacteriophages displaying antibodies differing in sequence (phage antibody library) was prepared by use of this technique, and an antibody binding to an arbitrary antigen can be selected (picked) from the library to obtain an antibody specifically binding to the desired antigen.
The phages used in the phage display technology are typically filamentous phages M13. The antibody display on phage particles can usually be carried out by inserting an antibody H chain variable region gene and L chain variable region gene linked to a gene encoding a phage coat protein such as g3p to phagemid vectors, and transferring the phagemid vectors to E. coli, which is then infected with a helper phage. For antibody screening from the phage antibody library, the antibody library is mixed with an immobilized antigen, and a phage displaying an antibody capable of binding to the antigen can be selected (picked) by binding, washing and elution procedures (panning). The recovered phage can be amplified by the infection of a host such as E. coli. The phage thus amplified can be used in repeated panning to thereby enhance the ratio of the antibody specifically binding to the antigen (Non Patent Literature 9).
In order to obtain an antibody fragment by the phage display method, an antibody library is usually prepared in the form of a fusion protein of Fab or single-chain Fv (scFv) and a phage coat protein. Although phage vectors containing the whole gene information of bacteriophages were initially used, current methods generally employ phagemid vectors. The phagemid vectors are plasmid vectors smaller in size than phage vectors. A gene encoding a protein to be displayed is linked to the end (which corresponds to the N terminus) of a gene encoding a phage coat protein, such as gene 3 or gene 8, and the resulting gene is inserted to phagemid vectors. In the phage display method, the gene encoding a protein to be displayed must be packaged in a phage particle. Therefore, a phage packaging signal needs to reside on the phagemid vectors. In addition, phage production from E. coli containing the phagemid vector requires infecting the E. coli with a helper phage, such as M13KO7 or VCSM13, which supplies a phage structural protein or the like.
Chain shuffling may be used as a method for identifying an antibody fragment having high affinity for a target antigen using the phage antibody library thus prepared. In this method, for example, a polynucleotide encoding an antigen-binding site (e.g., L chain variable region) of an antibody is diversified by random or site-directed mutagenesis, while a polynucleotide encoding another antigen-binding site (e.g., H chain variable region) of the antibody is fixed. This can be achieved, for example, by cloning a wild-type polynucleotide encoding the H chain variable region of an antibody binding to the target antigen, into a phage display vector system having a library of the diversified L chain variable region polynucleotides, and subsequently screening for an antibody binding with high affinity to the antigen. Typically, the H chain variable region is first fixed, while the L chain variable regions are shuffled. Examples of methods for affinity maturation of an antibody using such L chain shuffling may include: an approach using dual-vector system-III (DVS-III) composed of a set of a pLf1T-3 (L chain) phagemid vector and pHg3A-3 (H chain-gene 3) plasmid (Non Patent Literature 15); and an approach which involves carrying out panning operation for an antigen using a phage display library of H chain variable regions, and then carrying out panning operation again using the H chain variable regions thus enriched by panning operation in combination with VL genes in a library (Non Patent Literature 16).
The phage display method is also used as means to humanize a non-human animal-derived antibody binding to a target antigen. For example, human-derived antibody L chains are obtained by panning operation for an antigen using fixed H chains of an antibody obtained by mouse immunization and a human naive-derived L chain antibody library in combination. Subsequently, a human-derived antibody H chain can be further obtained by panning operation again for the antigen using the fixed L chains and a human naive-derived H chain antibody library in combination. In this way, a human antibody can be obtained on the basis of the non-human animal-derived antibody by the sequential replacement with the human antibody libraries (Non Patent Literature 17).
There are some reports on phage display modified by altering genes of helper phages. For example, Hyper phage (Non Patent Literature 10), CT helper phage (Non Patent Literature 11), and Ex-phage (Non Patent Literature 12) are known. The transfer of a gene encoding a substance inhibiting a drug resistance gene has been reported as an example of the transfer of a foreign gene to the genome of a bacteriophage (Non Patent Literature 13 and Patent Literature 10). However, none of the previous reports disclose the construction of a novel phage display method suitable for obtaining antibodies having common L chains or H chains by the alteration of a helper phage.